The Influence of Zinc on the Uptake and Loss of Cadmium and Lead in the Woodlouse, Porcellio scaber (Isopoda, Oniscidea)

Ecotoxicology and Environmental Safety 47, 43}53 (2000) Environmental Research, Section B doi:10.1006/eesa.2000.1940, available online at http://www.idealibrary.com on

The Influence of Zinc on the Uptake and Loss of Cadmium and Lead in the Woodlouse, Porcellio scaber (Isopoda, Oniscidea) Beate Witzel AG Bodenzoologie und OG kologie, Institut f uK r Biologie, Freie UniversitaK t Berlin, Grunewaldstrasse 34, D-12165 Berlin, Germany Received July 23, 1999

functions, the storage of metals in either one or both of these cell types and the characteristics of the metals themselves are important for the ability of the animals to eliminate them again. Cadmium, for example, is stored only in S cells, in the so-called &&cuprosomes'' and the cytosol, whereas zinc and lead can be stored in S cells as well as B cells. B cells have secretory function and void cell material in a 24-h rhythm into the lumen of the hepatopancreas, whereas S cells have only a storage function and never void any material. Thus metals that are stored in the granules of S cells remain in the body until the death of the animal (Hames and Hopkin, 1991a). Although there seems to be no point for further discussion contradictory statements have been made about the ability of P. scaber to eliminate cadmium. Since cadmium is stored mainly in S cells the animals should not be able to excrete cadmium once it has accumulated in the body. This hypothesis has been con"rmed in several papers (Hopkin et al., 1989b; Hopkin, 1990b; Witzel, 1998). But nevertheless in some studies the animals were able to excrete this metal (Hopkin, 1990a; Hames and Hopkin, 1991b). A consistent explanation of this e!ect is so far lacking. Assuming that this e!ect is caused by interaction of the metals and excluding lead by experience (Witzel, 1998), zinc was suspected to be responsible for changes in the storage of cadmium. Thus, this paper reports the results of feeding experiments with juvenile P. scaber designed to reveal the in#uence of zinc on the uptake and loss of lead and cadmium.

Uptake of cadmium, lead, and zinc was studied in juvenile Porcellio scaber in feeding experiments over 5 months. The metals were o4ered separately and in di4erent combinations and concentrations in the food. The ability of P. scaber to eliminate the accumulated metals was studied subsequently for 3 months on uncontaminated food. Characteristic patterns of accumulation are described for the three metals. The combination of lead and zinc resulted in only minor di4erences in these patterns. On the other hand, the combination of zinc and cadmium at high concentrations completely changed the accumulation patterns for both metals. Not only cadmium but also zinc was excreted by P. scaber exclusively when the animals had been contaminated with both metals. In contrast both metals were stored permanently when o4ered separately. Possible reasons for the interactions of cadmium and zinc are discussed.  2000 Academic Press Key Words: crustaceans; heavy metals; intermetallic e4ects; isopod.

INTRODUCTION

Since woodlice have been successfully adopted as monitor organisms for heavy metal pollution (Hopkin et al., 1986, 1989a, 1993; Dallinger et al., 1992; Witzel, 1992) many investigations have been carried out to study the mechanisms of uptake and storage of heavy metals in these animals. These studies, focused mainly on Porcellio scaber and Oniscus asellus, have revealed the processes of detoxifying heavy metals by storing them in a nontoxic form. As far as is known heavy metals are stored mainly in the hepatopancreas. This organ consists of B and S cells in which the metals are not only bound to speci"c low-molecular-weight compounds in the cytosol but are also stored in an undissolved nontoxic form in vesicles of lysosomal origin (for further information see Hopkin and Martin, 1982b, 1984; Hopkin, 1989, 1990b; Prosi and Dallinger 1988; Prosi et al., 1983; Donker et al., 1990). Because B and S cells have di!erent

MATERIALS AND METHODS

Animal Housing and Food Preparation Newly liberated juveniles of P. scaber Latreille, 1804, were fed contaminated leaves of hornbeam (Carpinus betulus) for 5 months. These juveniles were taken from a laboratory stock culture from which gravid females were separated to collect the young. Animals were kept in plastic boxes (13;18;6.5 cm) with a layer of arti"cial soil and a perforated lid. During the experiment animals were fed a de"ned

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0147-6513/00 $35.00 Copyright  2000 by Academic Press All rights of reproduction in any form reserved.

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TABLE 1 Heavy Metal Concentrations and Combinations of Metals in Contaminated Leaf Litter Concentration I Cd-alone Pb-alone Zn-alone Cd/Zn Pb/Zn Cd/Pb/Zn

3.0 lg g\ 100 lg g\ 200 lg g\ 3.0/200 lg g\ 100/200 lg g\ 3.0/100/200 lg g\

Concentration II 0.027 lmol g\ 0.48 lmol g\ 3 lmol g\

amount of leaves every month. The boxes were placed in a climatized room at 183C with 12 h dimmed light day\ and were moistened every 3 days with deionized water. Contaminated leaves were prepared by soaking in solutions of cadmium chloride, lead chloride, and zinc chloride for 3 h and dried for 24 h at 503C before weighing. The concentrations in the leaves were monitored using heavy metal analyses and were found to be within the expected range of reproducibility.

30 lg g\ 1000 lg g\ 2000 lg g\ 30/2000 lg g\ 1000/2000 lg g\ 30/1000/2000 lg g\

0.266 lmol g\ 4.8 lmol g\ 30.6 lmol g\

Statistics The data were "rst tested for normality. Data from the period of clearance were subjected to a two-way analysis of variance (ANOVA) using the factors time and metal content. When the ANOVA revealed di!erences in metal content, a Student t test was used to de"ne the time interval when metals were excreted. A probability of error (P) of 5% was chosen as the level of signi"cance.

Exposure During the period of exposure to contaminants (here called contamination, see Witzel, 1998) animals were fed leaves with two concentrations and di!erent combinations of heavy metals (Table 1). There were two replicates for each metal concentration and combination, with 240 animals of the same age in each box. Six control boxes were furnished with uncontaminated leaves. The natural background contamination in the leaves was 10 lg g\ Pb, 0.3 lg g\ Cd, and 100 lg g\ Zn. During the experiment the concentrations of cadmium, lead, and zinc in the animals were monitored. Every 4 weeks 10 animals per box were randomly selected and kept on wet blotting paper for 24 h to allow defecation before metal analysis. The animals were individually placed in microcups, frozen, dried, weighed, and analyzed for zinc, cadmium, and lead by #ame or #ameless atomic absorption spectrometry using the method described by Witzel (1998). The data on the replicates were pooled. Since the juveniles were growing throughout the experiments the results are expressed as metal content per animal on a dry weight basis (ng/animal) to eliminate dilution bias.

RESULTS

Three patterns of metal accumulation can be distinguished in relation to the three metals tested in this study. The "rst type is characterized by cadmium with a constant concentration factor of about 10 (cf"animal concentration/food concentration) in all concentrations, steady accumulation during contamination up to Week 20, and no decrease in content on clean food. The metal content increased even on uncontaminated food, indicating that the uptake continued (Fig. 1).

Clearance After 5 months on contaminated leaves the remaining animals in each box were kept on wet blotting paper for 2 days and then transferred to new boxes with uncontaminated leaves. Subsequently the elimination of metals (here called decontamination) was studied by heavy metal analysis of 6}10 animals per box after 1, 2, 4, 6, 8, and 12 weeks of feeding on uncontaminated litter. The control animals were again analyzed every 4 weeks.

FIG. 1. Cadmium content in P. scaber during the period of contamination and decontamination in the experiment: Cd-alone, conc. I and II. Means$SEM.

UPTAKE AND LOSS OF Zn, Cd, Pb, IN Porcellio scaber

The second pattern is represented by lead. Speci"c characteristics are a low cf of 0.2 for concentration (conc.) I and conc. II up to 0.5 in the control and an immediate drop in content on clean food of about 40}60%. At the end of decontamination the animals receiving conc. I reached the content of the control, while in those receiving conc. II, the lead content remained elevated (Fig. 2, Table 2). The third pattern is represented by zinc. The cf is low on contaminated food (about 0.6) and high on clean food (about 6 in Week 32 for all concentrations). Di!erent phases of accumulation can be distinguished: "rst, an increase in content up to Week 20 (phase A); second, a plateau up to Weeks 24/26 (phase B); and "nally, a rapid increase in content up to Week 32 (phase C). In the control only two phases can be discriminated (Fig. 3); phase B is lacking. Animal growth also indicates two phases, but only for the contaminated animals (Fig. 4). Here growth of animals consuming contaminated food decreased, but after these animals fed on uncontaminated litter, growth increased instantly. In the control, growth is linear; nevertheless, zinc

FIG. 2. Lead content in P. scaber during the period of contamination and decontamination in the experiment: (a) Pb-alone, conc. I; (b) Pb-alone, conc. II. Means$SEM.

45

uptake follows two phases. It is to be assumed that the uptake of zinc follows an ontogenetic need that increases from Week 24 onward. Therefore, at the end of the experiment the animals fed conc. I and conc. II reached the same zinc load of about 1800 ng ind\. To compare the uptake of zinc between the di!erent experiments linear regression lines for the phases A and C are used (Table 3). When zinc and lead were combined the accumulation characteristics of the metals were only slightly a!ected for zinc (Figs. 5, 6). Signi"cant di!erences were found at higher zinc concentrations and contents in Week 20 compared with zinc-alone, indicating increased zinc uptake in the presence of lead at conc. II. The lower lead content in Week 20 (conc. II), on the other hand, is caused only by the lower average weight of the animals in this experiment compared with the lead-alone experiment (see Table 4). The regression lines for zinc con"rm higher zinc uptake on contaminated food but lower uptake on clean food compared with zinc-alone. The combination of zinc and cadmium a!ects the characteristics of both metals at conc. II (see Figs. 7b, 8). Zinc exhibited higher uptake during contamination, leading to signi"cantly higher concentrations by Week 20 compared with zinc-alone (see Tables 2, 4). In the highly contaminated animals the characteristic phases B and C for zinc uptake on uncontaminated litter have completely changed. At conc. II the high increase in zinc content up to Week 28 was followed by a signi"cant loss of zinc by Week 32. Zinc uptake at conc. I was not a!ected by cadmium. For cadmium at conc. II two declines in content can be described during decontamination: one from Week 20 to Week 21, followed by another uptake, and a second one from Week 28 to Week 32, simultaneously with the excretion of zinc. Sample animals were slightly but not signi"cantly lighter in Week 32 than in Week 28 (Table 5). Thus, the weight of the animals was not responsible for the signi"cantly reduced metal content in Week 32. Cadmium was excreted when the animals had reached a body burden of 540 ng (4.8 nmol). Like zinc, cadmium accumulated to a greater degree up to Week 20 compared with cadmium-alone, leading to signi"cantly higher concentrations (see Tables 2, 4). Uptake of cadmium at conc. I was not a!ected by the presence of zinc (Fig. 7a). The changes in contents during decontamination were not signi"cantly di!erent. The results of the Zn/Cd/Pb experiment con"rm the changes in zinc and cadmium when these metals are combined in high concentrations. Again, uptake and loss of lead are not in#uenced by the presence of zinc (Fig. 9). But as for the Zn/Cd experiment, there was a signi"cant loss of cadmium from Week 28 to Week 32 at conc. II (Fig. 10b) when the animals had reached a body burden of 450 ng (4 nmol). Accumulation of cadmium during contamination was not elevated compared with cadmium-alone. Uptake of cadmium at conc. I was not a!ected; there was no loss of

46

TABLE 2 Results of the Feeding Experiments in P. scaber with Cd-Alone, Pb-Alone, and Zn-Alone in the Control, and at Concentrations I and II? Cadmium Control Cd Conc. I Cd Conc. II (mg dw) (lg/g) (mg dw) (lg/g) (mg dw)

Week 4 8 12

20 21 22 24 26 28 32

Mean SD Mean SD Mean SD Mean SD Mean SD Mean SD Mean SD Mean SD Mean SD Mean SD Mean SD

?dw, dry weight.

0.25 0.04 0.49 0.10 0.73 0.11 1.06 0.19 1.30 0.39

1.53 0.44 1.45 0.36 1.57 0.18 2.03 0.44 1.96 0.25

1.56 0.28

2.61 0.39

1.85 0.36 2.50 0.33

2.93 0.38 5.34 0.98

0.186 0.032 0.377 0.076 0.622 0.101 0.958 0.169 0.900 0.145 1.135 0.193 1.124 0.213 1.320 0.381 1.587 0.353 1.952 0.382 2.385 0.465

13.82 2.93 17.97 3.82 24.39 2.84 35.40 4.10 45.56 5.84 38.91 6.46 36.36 7.86 32.86 6.09 30.30 6.33 26.02 4.38 19.35 4.08

0.201 0.037 0.281 0.049 0.550 0.109 0.758 0.145 0.868 0.151 0.837 0.245 1.009 0.312 1.044 0.232 1.106 0.272 1.265 0.252 1.736 0.347

Cd (lg/g) 136.92 23.35 179.56 39.71 249.10 55.22 284.14 47.77 409.16 61.47 391.76 53.37 373.19 70.32 339.32 67.98 314.55 46.51 279.60 66.49 234.46 51.17

Zinc

Control Pb Conc I Pb Conc.II (mg dw) (lg/g) (mg dw) (lg/g) (mg dw) 0.342 0.061 0.689 0.105 0.966 0.227 1.334 0.329 2.018 0.709

2.8 1.0 4.5 2.0 5.1 1.8 4.4 1.7 2.6 0.8

2.234 0.645

3.3 1.0

2.382 0.524 3.335 0.502

2.9 0.7 2.8 1.3

0.190 0.026 0.411 0.073 0.503 0.104 0.798 0.201 0.794 0.152 0.961 0.226 1.137 0.239 1.174 0.164 1.163 0.379 1.734 0.378 1.921 0.383

19.9 4.5 25.0 7.9 22.4 7.0 26.1 6.6 23.8 9.1 15.5 5.5 10.5 2.8 9.3 3.8 6.1 1.9 6.0 2.3 4.2 2.5

0.203 0.029 0.454 0.086 0.588 0.102 0.931 0.189 0.976 0.230 1.223 0.349 1.356 0.392 1.383 0.295 1.542 0.304 1.724 0.375 2.110 0.498

Pb (lg/g)

Control (mg dw)

Zn Conc. I Zn Conc. II (lg/g) (mg dw) (lg/g) (mg dw)

243.0 66.1 316.4 69.1 428.9 122.1 330.3 85.2 343.0 95.7 192.9 65.8 194.2 77.5 148.1 64.8 119.6 40.2 106.5 42.9 73.0 26.0

0.316 0.061 0.430 0.063 0.698 0.130 1.361 0.297 1.749 0.373

257.2 176.4 286.7 178.0 376.7 84.2 406.8 85.8 357.8 83.7

2.613 0.764

306.1 91.0

3.598 0.512 3.624 0.652

552.4 106.6 655.6 122.4

0.476 0.070 0.787 0.122 1.021 0.184 1.014 0.196 1.183 0.236 1.263 0.244 1.431 0.228 1.725 0.440 1.850 0.308 2.955 0.851

235.5 61.3 292.4 64.8 315.6 105.6 436.5 169.0 413.1 133.9 358.3 55.6 388.4 119.7 396.8 97.6 608.1 125.7 572.5 118.1

0.318 0.080 0.608 0.104 0.725 0.163 0.740 0.140 0.937 0.203 1.026 0.373 1.191 0.246 1.566 0.431 1.849 0.573 2.581 0.523

Zn (lg/g)

577.4 180.0 815.2 230.3 954.6 290.6 1158.4 340.3 1018.1 230.7 954.8 344.3 769.9 263.4 774.6 267.4 801.9 197.1 785.8 225.5

BEATE WITZEL

16

Lead

47

UPTAKE AND LOSS OF Zn, Cd, Pb, IN Porcellio scaber

TABLE 3 Parameters of Regression Lines for the Uptake of Zinc at Concentrations I and II by P. scaber in Phase A (Contamination) and Phase C (Decontamination) Phase A Experiment Zn I Zn II Zn/Pb I Zn/Pb II Zn/Cd I Zn/Cd II Zn/Cd/Pb I Zn/Cd/Pb II FIG. 3. Zinc content in P. scaber during the period of contamination and decontamination in the experiment: Zn-alone, conc. I and conc. II. Means$SEM.

cadmium on clean food and changes in content were not signi"cant (Fig. 10a). At conc. II there were two declines in zinc: one from Week 20 to Week 22, and another from Week 28 to Week 32, the latter being simultaneous with cadmium (Fig. 11). On the contrary, lead content was not signi"cantly reduced in this period. As before, uptake of zinc at conc. I is not a!ected by the presence of the other metals. Again, uptake of zinc on contaminated food at conc. II was elevated by the presence of cadmium and lead, leading to higher concentrations in Week 20 compared with zinc-alone (see Tables 2, 5). Table 6 provides an overview of the molar concentrations of the animals during the period of decontamination. Obviously the excretion of zinc and cadmium was not induced by

FIG. 4. Growth of P. scaber during the period of contamination and decontamination in the experiment: Zn-alone, control, conc. I and conc. II. Means (dry weight)$SEM.

a

b

!102.50 26.98 !218.18 54.72 !60.87 31.61 !250.97 71.27 !41.90 26.95 !732.96 149.85 13.60 16.14 !459.18 96.61

Phase C R 0.998 0.988 0.998 0.989 0.988 0.971 0.994 0.926*

a

b

!3120.2 150.27 !2366.2 136.74 !1723.7 96.87 !858.7 89.85 !3634.0 196.26

r 0.987 0.998 0.982* 0.989 0.956**

>"a#b * x; all r values are signi"cant at P40.01 except *P"0.02 and **P"0.04.

saturation of the storage capacity for these metals. The molar body burden of the animals in the experiments was quite di!erent.

FIG. 5. Lead content in P. scaber during the period of contamination and decontamination in the experiment: (a) Pb/Zn, conc. I; (b) Pb/Zn, conc. II. Means$SEM.

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BEATE WITZEL

FIG. 6. Zinc content in P. scaber during the period of contamination and decontamination in the experiment: Pb/Zn conc. I and conc. II. Means$SEM.

DISCUSSION

The chosen exposure levels for zinc, cadmium, and lead lie within the range of contamination found in the "eld. Concentration II represents heavily contaminated conditions but did not lead to refusal of food by the animals although

growth was reduced on contaminated food. Critical concentrations for food acceptance are 54 lmol g\ resp. 46 lmol g\ Zn (Donker et al., 1996; Joosse et al., 1983). Hopkin and Hames (1994) found critical food concentrations of 100 lg Cd g\, 2000 lg Pb g\, and 1000 lg Zn g\; food containing 2000 lg Zn g\ was de"nitely toxic, causing the death of the animals within 30 days. Hopkin (1990) found that animals die of zinc poisoning at body concentrations of 800 lg Zn g\. In the present study all animals at conc. II reached a whole-body concentration of more than 1000 lg Zn g\ in Week 20, and in the Zn/Cd and the Zn/Cd/Pb exposures they had concentrations greater than 2000 lg Zn g\ but no toxic e!ects were observed. Mortality was similar in all experiments. The characteristics described for lead are similar to the results of Hopkin (1990a), Hopkin and Hames (1994), and Witzel (1998). Initial elimination of lead is very e!ective and reduces lead content to about 30% within 1 week. The animals can eliminate the assimilated lead to about 50%, but after 2}4 weeks of feeding on uncontaminated litter the decline ceased (see Witzel, 1998). According to the histochemical "ndings of, e.g., Hopkin and Martin (1982b) and Hames and Hopkin (1991a) lead is stored in B and S cells. The lead remaining after decontamination probably re#ects the amount of lead stored in S cells. Uptake and loss of lead

TABLE 4 Results of the Feeding Experiments in P. scaber with Pb/Zn and Cd/Zn at Concentrations I and II? Pb/Zn

Week 4 8 12 16 20 21 22 24 26 28 32

Mean SD Mean SD Mean SD Mean SD Mean SD Mean SD Mean SD Mean SD Mean SD Mean SD Mean SD

? dw, dry weight.

Cd/Zn

Conc. I (mg dw)

Pb (lg/g)

Zn (lg/g)

Conc. II (mg dw)

Pb (lg/g)

Zn (lg/g)

Conc. I (mg dw)

Cd (lg/g)

Zn (lg/g)

Conc. II (mg dw)

Cd (lg/g)

Zn (lg/g)

0.247 0.042 0.528 0.083 0.741 0.297 0.798 0.248 0.999 0.182 1.097 0.339 1.398 0.565 1.279 0.432 1.723 0.461 1.876 0.463 2.405 0.604

23.3 7.3 20.8 5.7 21.7 4.9 19.4 3.1 21.7 5.8 12.6 4.2 10.1 2.3 8.3 2.4 8.4 3.1 5.4 2.1 4.3 1.6

264.7 107.4 350.6 71.1 475.4 146.6 586.0 178.1 560.2 173.0 607.5 185.5 481.6 124.1 447.8 165.0 536.9 188.3 528.5 154.6 576.1 155.5

0.204 0.050 0.512 0.082 0.495 0.098 0.622 0.171 0.639 0.163 0.858 0.275 1.088 0.477 1.407 0.340 1.935 0.536 2.088 0.561 3.321 0.962

120.3 31.6 218.1 64.5 281.5 67.8 244.2 63.3 355.8 126.6 206.7 48.4 150.2 40.7 100.7 35.9 81.0 30.6 60.2 22.3 45.5 14.9

333.7 107.9 699.5 185.0 1005.8 232.4 1464.6 395.9 1950.0 566.4 1732.3 499.7 1134.0 317.4 926.9 261.4 800.0 236.5 766.3 203.1 644.1 201.9

0.328 0.050 0.609 0.103 0.708 0.111 0.789 0.155 1.056 0.194 1.725 0.274 1.808 0.344 1.769 0.290 2.494 0.603 2.574 0.421 3.468 0.412

16.91 3.92 19.83 3.53 36.51 6.76 51.66 12.61 51.86 14.84 44.66 8.94 45.97 9.76 32.44 5.95 21.41 2.59 26.986 4.792 23.58 2.80

240.4 55.2 221.1 79.2 440.9 102.2 503.7 111.8 476.7 189.0 473.6 133.6 486.7 92.6 504.1 158.7 686.2 120.1 675.4 116.5 769.7 192.6

0.232 0.046 0.442 0.066 0.779 0.155 0.937 0.217 1.145 0.378 1.269 0.266 1.223 0.424 1.446 0.189 1.988 0.336 2.278 0.425 2.134 0.425

89.30 26.29 148.98 32.50 256.79 26.00 297.72 28.12 488.03 87.66 330.15 30.58 334.03 73.07 314.32 59.81 268.98 66.99 249.61 45.43 168.48 30.98

341.6 147.4 587.3 113.6 1441.0 175.0 1503.0 378.6 2259.4 549.0 2123.8 597.3 2067.7 353.1 1565.3 457.3 1244.5 232.9 1384.9 336.8 1169.6 195.4

UPTAKE AND LOSS OF Zn, Cd, Pb, IN Porcellio scaber

FIG. 7. Cadmium content in P. scaber during the period of contamination and decontamination in the experiment: (a) Cd/Zn, conc. I; (b) Cd/Zn, conc. II. Means$SEM.

were not signi"cantly in#uenced by the presence of zinc, although there were di!erences in the lead content in the di!erent experiments. But this e!ect was more likely caused

FIG. 8. Zinc content in P. scaber during the period of contamination and decontamination in the experiment: (a) Cd/Zn conc. I and conc. II. Means$SEM.

49

by di!erences in the weight of the animals. Based on an average body weight in all experiments the resulting lead contents indicate no di!erences. Although lead and zinc underlie the same chemical reactions and are stored in the same cells (see Hopkin, 1990b) no antagonistic e!ects were observed within the chosen concentrations. The characteristics of cadmium were described by Witzel (1998) and are now con"rmed by the Cd-alone experiment in this study. As described in the histochemical papers of Hopkin et al. (1989a) and Hames and Hopkin (1991a), cadmium is accumulated permanently up to the death of the organism. These authors postulated that cadmium cannot by excreted by P. scaber once it is assimilated and stored. Cadmium is stored up to 80% in the hepatopancreas of P. scaber and here it occurs only in S cells (Dallinger and Prosi, 1988). But S cells have only a storage function and never exhibit any loss of cell material. Cadmium was found not only in the &&copper granules'' of the cells but also in the cytoplasm bound to a speci"c low-molecular-weight compound (Dallinger and Prosi, 1988; Donker et al., 1990). Dallinger (1993) postulated two cadmium pools in the cells: immobile cadmium in the lysosomes and a mobile fraction in the cytosol, which can be excreted at low rates. Aging cells at the proximal end of the hepatopancreas may void their content when breaking down, but this amount may be negligible. In contrast to these "ndings the present study reveals signi"cant excretion of cadmium when combined with zinc in high concentrations. Interestingly, the losses of cadmium and zinc both occurred either between Weeks 20 and 22 or between Weeks 28 and 32. The resulting conclusion is that the presence of zinc interferes with the storage of cadmium in S cells at high concentrations. Therefore, uptake of cadmium does not result in permanent storage. Only when combined with zinc at high concentrations can cadmium be excreted separately or simultaneously with zinc. These results are supported by data from Hopkin (1990a) and Hames and Hopkin (1991b) where P. scaber, contaminated with zinc and cadmium, demonstrated a loss of cadmium when feeding on clean food. Donker (1992) found a negative correlation between cadmium and zinc in the hepatopancreas. The more zinc that was stored, the less cadmium that was found in this organ down to 42%. The rest of the cadmium was found in the remainder of the body. Based on these "ndings Donker suggests a limited storage capacity for metals in the hepatopancreas. However, this assumption is not supported by the present study. The levels of contamination at which elimination of cadmium occurred were quite di!erent between experiments. The fact that cadmium is not excreted when animals are contaminated with this metal alone or in combination with lead (Witzel, 1998) contradicts the assumption of a &&mobile fraction'' of cadmium in S cells (Dallinger, 1993). More likely this metal

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TABLE 5 Results of the Feeding Experiments in P. scaber with Cd/Pb/Zn at Concentrations I and II? Conc. I

Week 4 8 12 16 20 21 22 24 26 28 32

Mean SD Mean SD Mean SD Mean SD Mean SD Mean SD Mean SD Mean SD Mean SD Mean SD Mean SD

Conc. II

Conc. I (mg dw)

Cd (lg/g)

Pb (lg/g)

Zn (lg/g)

Conc. II (mg dw)

Cd (lg/g)

Pb (lg/g)

Zn (lg/g)

0.339 0.078 0.455 0.073 0.531 0.119 0.635 0.182 0.762 0.186 1.345 0.212 1.354 0.333 1.407 0.270 1.452 0.401 1.719 0.278 2.676 0.771

10.76 3.77 23.60 5.22 35.97 6.41 50.94 8.90 64.26 16.85 48.69 5.81 71.16 30.28 42.66 10.42 38.00 6.01 38.29 7.75 26.84 5.18

13.6 7.1 11.2 2.9 14.6 4.1 19.5 4.9 27.3 10.1 9.3 1.9 11.3 3.2 5.2 1.2 4.8 1.5 4.2 0.8 3.7 1.0

264.9 70.2 311.2 71.0 362.3 112.4 410.6 143.3 496.0 260.6 520.3 126.3 586.5 185.8 533.4 158.6 416.0 97.7 411.1 83.1 592.6 142.9

0.264 0.034 0.391 0.092 0.476 0.146 0.551 0.163 0.826 0.266 1.209 0.310 1.327 0.299 1.359 0.175 1.417 0.290 1.689 0.415 1.684 0.312

60.71 14.14 131.01 23.23 212.70 47.88 290.61 46.35 397.48 55.06 302.50 72.95 252.20 54.18 261.55 41.12 253.85 59.20 258.05 41.26 175.01 38.13

71.4 22.4 228.3 75.8 250.4 62.5 266.9 60.3 272.1 70.4 189.5 72.2 153.8 69.4 140.7 54.6 90.6 25.4 90.9 46.1 57.1 19.2

392.9 52.2 687.2 188.2 1260.4 324.5 1389.3 411.6 2248.0 562.0 1406.1 521.6 851.7 322.4 895.6 165.2 883.6 258.6 1089.7 203.2 895.0 245.5

? dw, dry weight.

can be eliminated only when its storage in S cells is disrupted by zinc. In contrast to lead and cadmium, zinc is an essential metal, e.g., in the enzyme carbonic anhydrase. The uptake of zinc has to deal with the physiological need for this metal on the one hand and the avoidance of a surplus of zinc under contaminating conditions on the other hand. Thus, net zinc uptake has to be regulated to provide the animal with enough zinc without exhausting immediately the storage capacity (see Joosse et al., 1981). Dallinger (1993) described P. scaber as a facultative concentrator for zinc. In the literature the concentration factors vary from 0.04 to 7, representing a wide range of regulatory capacity in zinc assimilation. In the present investigation the regulation of zinc uptake is re#ected by 10-fold higher concentration factors on clean food compared with contaminated food. Thus, zinc uptake was negatively correlated with food concentration. On changing to uncontaminated food, net uptake of zinc ceased for 2 to 4 weeks but then increased rapidly. This phase of constant contents may re#ect the time the animals needed to adapt their assimilation e$ciency to the low zinc concentration of clean food. Contaminated animals at conc. I and conc. II contained the same amount of zinc at the end of the investigation, indicating a physiological need for zinc with age or size. The uptake of zinc always followed a linear

regression. An exponential increase in zinc content, like Hopkin and Martin (1984) described for Oniscus asellus on contaminated litter, was never observed. In this study characteristic phases of zinc uptake are described. The presence of lead a!ected only the rate of uptake, i.e., the regression slope of these phases. But when combined with cadmium, the characteristics of zinc were mostly changed. This is di!erent from the results of Donker and Bogert (1991) and Hopkin and Hames (1994), who did not "nd intermetallic e!ects on the uptake of zinc. On the contrary, Hopkin and Martin (1984) and Tomita et al. (1992) found that lead contamination increases zinc uptake in woodlice as in the present study. Like cadmium, zinc is stored mainly in the hepatopancreas (Hopkin, 1990a). In contaminated areas this organ is enlarged, containing up to 80% of the zinc content, and contaminated animals stored relatively more zinc in this organ than did uncontaminated animals (Hopkin and Martin, 1982a; Donker, 1992). Theoretically, zinc is stored in B and S cells. But the ability of P. scaber to excrete zinc varies. Contaminated animals feeding on uncontaminated litter exhibited no loss of zinc (Hopkin, 1990a; Donker et al., 1996) or excreted zinc to various degrees (Hames and Hopkin, 1991b; Bibic et al., 1997). Assuming that zinc can be excreted from B cells or the rest of the body, no elimination

UPTAKE AND LOSS OF Zn, Cd, Pb, IN Porcellio scaber

51

FIG. 9. Lead content in P. scaber during the period of contamination and decontamination in the experiment: (a) Cd/Pb/Zn, conc. I; (b) Cd/Pb/Zn, conc. II. Means$SEM.

FIG. 10. Cadmium content in P. scaber during the period of contamination and decontamination in the experiment: (a) Cd/Pb/Zn, conc. I; (b) Cd/Pb/Zn, conc. II. Means$SEM.

is detected when it is stored only in S cells. In this study zinc was excreted exclusively after combination with cadmium at high concentrations, while there was no e!ect at conc. I, representing moderate contamination. The level of contamination at which excretion started was di!erent in the experiments. In the Zn/Cd experiment, zinc was excreted after reaching 3000 ng ind.\ (37 nmol ind.\), while in the Zn/Cd/Pb experiment, excretion started at 1700 ng (27 nmol ind.\). As for cadmium, a main conclusion can be drawn: The presence of lead and cadmium at high concentrations results in increased zinc uptake. Only after combined contamination with cadmium at high concentrations was zinc excreted. No critical level of contamination that induces the elimination of zinc could be detected. These "ndings indicate that combined uptake of zinc and cadmium does not result in the permanent storage of these metals in the hepatopancreas. Although Donker (1992) found that the more zinc is stored in the hepatopancreas, the less cadmium is stored, the present results suggest that both metals impede each other in their storage in S cells. Donker

et al. (1996) found that zinc uptake "rst culminates in increasing concentrations in remaining tissues of P. scaber, followed by translocation of zinc into the hepatopancreas.

FIG. 11. Zinc content in P. scaber during the period of contamination and decontamination in the experiment: Cd/Pb/Zn, conc. I and conc. II. Means$SEM.

52

BEATE WITZEL

TABLE 6 Molar Content of Heavy Metals in P. scaber during Decontamination? Content (nmol animal\)

Zn/Cd/PbII Cd Pb Zn Zn/Cd II Cd Zn Zn/Pb II Pb Zn

Week 20

Week 21

Week 22

Week 24

Week 26

Week 28

Week 32

3.15 1.48 17.11

3.14 1.10 25.37

2.92 0.94 16.88

3.18 0.93 18.63

3.18 0.63 18.70

4.02 0.78 30.86

2.58 0.47 22.07

4.79 38.17

3.73 39.76

3.64 37.13

4.00 34.15

4.71 37.79

5.08 47.69

3.13 37.23

1.14 21.18

0.79 22.01

0.78 17.60

0.74 20.00

0.73 24.80

0.52 25.90

0.70 33.90

? For SD see Tables 2, 4, and 5.

They postulated that zinc can be excreted by urinary excretion immediately after uptake before it is stored in the hepatopancreas. This would explain the reduced net uptake of zinc-adapted populations of P. scaber. But in this study zinc was not eliminated immediately after uptake. The zinc content of the animals increased continually. The present results indicate that combined contamination with cadmium and zinc inhibits the complete translocation of the assimilated metals into the hepatopancreas. A fraction of zinc and/or cadmium remains in the hemolymph or in other tissues. From there the metals can be excreted. Saturation of S cells as a reason for incomplete storage is not assumed. Uptake of zinc and cadmium was lower in the Zn/Cd/Pb experiment than in the Zn/Cd experiment. Whether this di!erence was caused by the presence of lead remains uncertain. CONCLUSION

The uptake and loss of cadmium and zinc in P. scaber are a!ected by the presence of other heavy metals. This study demonstrated that P. scaber is able to excrete cadmium and zinc only after contamination with both metals. The presence of lead enhances the uptake of zinc. The present results provide for a better understanding of the heavy metal physiology of a common test organism. Since P. scaber is frequently used for active or passive monitoring of heavy metals these interactions of metals have to be taken into account. ACKNOWLEDGMENTS The author thanks Gerd Weigmann and Gerhard Scholtz for fruitful discussions on the manuscript and Colin McLay for correcting and improving the English. This study was supported by a grant of the Free University Berlin.

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